System for analyzing tissue perfusion using concentration of indocyanine green in blood

ABSTRACT

The present invention relates, in general, to a system for analyzing tissue perfusion using the concentration of indocyanine green and a method of measuring the perfusion rate using the system and, more particularly, to a system for measuring tissue perfusion by injecting indocyanine green into a living body, detecting variation in the concentration of indocyanine green with the passage of time, and analyzing the detected variation, and a method of measuring the perfusion rate using the system. The present invention provides a method of measuring perfusion in a living body, which enables accurate measurement for respective regions in a wide range from a perfusion rate decreased to less than 10% of normal perfusion to a perfusion rate increased to greater than normal perfusion using the above-described mechanism of ICG in a living body, which cannot be conducted using the conventional technology.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national stage filing ofInternational Application No. PCT/KR2007/003355 filed on Jul. 11, 2007,which claims priority to, and the benefit of, Korean Patent ApplicationNo. 10-2006-0099033 filed on Oct. 11, 2006. The contents of theaforementioned applications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a system for analyzing tissue perfusionand a method of measuring the perfusion rate using the system. Moreparticularly, the present invention relates to a system for measuringtissue perfusion by injecting indocyanine green into a living body,detecting variation in the concentration of indocyanine green with thepassage of time, and analyzing the detected variation, and a method ofmeasuring the perfusion rate using the system.

BACKGROUND

The conventional tissue perfusion measuring method ‘laser Dopplerimaging’ is a method of measuring the degree of scattering of laserlight depending on the speed of blood flow in a skin surface, but has ashortcoming in that it is unsuitable for a method of measuring variationin the state in which blood flow is low because sensitivity is low whenblood flow rate decreases to less than 20% of the normal of the bloodflow rate.

Another conventional blood vessel imaging method ‘X-ray blood vesselimaging (X-ray angiography)’ shows X-ray images using a blood vesselcontrast agent. However, it is a structural imaging technique that showsthe configuration of the internal diameters of blood vessels, ratherthan the actual flow of blood (Helisch, A., Wagner, S., Khan, N.,Drinane, M., Wolfram, S., Heil, M., Ziegelhoeffer, T., Brandt, U.,Pearlman, J. D., Swartz, H. M. & Schaper, W. (2006) Arterioscler.Thromb. Vasc. Biol., 26: 520-526). Accordingly, it is currentlyimpossible to clinically measure the precise rate of tissue perfusionusing this method.

The safety of a conventional blood vessel imaging method usingindocyanine green (ICG angiography) has already been verified (Sekimoto,M., Fukui, M. & Fujita, K. (1997) Anaesthesia 52: 1166-1172), and themethod has been clinically used for the detection of the formation ofblood vessels in grafted skin (Holm, C., Mayr, M., Hofter, E., Becker,A., Pfeiffer, U. J. & Muhlbauer, W. (2002) Br. J. Plast. Surg. 55:635-644) and the measurement of newly created blood vessels in adiabetic patient's eyeballs (Costa, R. A., Calucci, D., Teixeira, L. F.,Cardillo, J. A. & Bonomo, P. P. (2003) Am. J. Opthalmol. 135: 857-866).ICG receives near infrared rays, having wavelengths ranging from 750-790nm, and radiates near infrared rays, having longer wavelengths rangingfrom 800 to 850 nm, and the radiated near infrared rays can be measuredusing a CCD camera or a spectrometer. Near infrared rays have highpenetration power, and thus they can penetrate several centimeters deepinto tissue and don't scatter much, with the result that they are theobject of extensive research towards human body imaging technology(Morgan, N. Y., English, S., Chen, W., Chernomordik, V., Russo, A.,Smith, P. D. & Gandjbakhche, A. (2005) Acad. Radiol. 12: 313-323). Thismethod is also used as a technique for structural blood vessel imaging.This method is used for the test of the permeability of newly createdblood vessels in a diabetic patient's eyeballs, not for the measurementof tissue perfusion.

The above method is used for an ‘ICG elimination test’ as well as theabove purpose. When ICG is injected into a vein, ICG is attached toprotein in a blood vessel, such as albumin, and is rapidly spread intothe body via blood vessels. When it is transferred to the liver, it isseparated from protein and discharged in the form of bile and excretedfrom the body finally while the protein is degraded. As a result, theconcentration of ICG is rapidly reduced in the blood vessels, so thatthe intensity of ICG fluorescence signals is reduced to half of theintensity of initial ICG fluorescence signals 4 to 6 minutes later, andis then diminished and deviates from an accurate measurement range. Oneuse of the ‘ICG dynamics’, in which ICG is rapidly eliminated by theliver, is a liver function test (Sinyoung Kim, et al., (2003) Kor. J.Lab. Med., 23: 88-91), but the ICG dynamics has been known to haveshortcomings when applied to in vivo imaging (Sekimoto, M., Fukui, M. &Fujita, K. (1997) Anaesthesia 52: 1166-1172).Accordingly, the presentinventors have completed the present invention through the ascertainmentof the fact that accurate measurement can be conducted in a wide rangefrom a perfusion rate decreased to less than 10% of that of normalperfusion to a perfusion rate increased to greater than that of normalperfusion using the ICG dynamics in the living body.

DISCLOSURE Technical Problem

An object of the present invention is to provide a method of measuringperfusion in a living body, which enables to provide absolute value ofperfusion for respective regions in a wide range from a perfusion ratedecreased to less than 10% of normal perfusion to a perfusion rateincreased to greater than normal perfusion using the above-described ICGdynamics in a living body, which cannot be conducted in the prior art.

Technical Solution

In order to accomplish the above object, the present invention providesa tissue perfusion analysis apparatus, a method of measuring tissueperfusion and a tissue perfusion measurement apparatus, which calculatea perfusion rate by injecting indocyanine green into a living body andmeasuring variation in the concentration of the indocyanine green.

DESCRIPTION OF DRAWINGS

FIG. 1 is a photo showing the fact that there is no correlation betweenlaser Doppler images and the rates of tissue necrosis.

FIG. 2 is a schematic diagram showing the overall construction fordetecting ICG fluorescence signals:

Description of reference numerals of principal elements in theaccompanying drawings:

1: light source, 2: living body stand,

3: 800˜850 nm filter, 4: ICG near infrared ray fluorescence detector,and

5: ICG image analysis apparatus.

FIG. 3 is a graph (upper side) showing the experimental results of thedynamics of the intensity of ICG fluorescence, directly acquired withthe passage of time from the blood in a living body, and a photo (lowerside) showing the results of analysis of indocyanine fluorescence.

FIG. 4 is a simulation graph showing the mechanism of ICG fluorescencebased on the perfusion rates of ischemic tissue.

FIG. 5 is a simulation graph showing the relationship between T_(max)and perfusion rates.

FIG. 6 is an imaging view of a blood vessel acquired after the injectionof ICG into the tail vein of a nude mouse.

FIG. 7 is an image outputted by mapping perfusion rates as colors, usingan ischemia pattern analysis method in a mouse from which a thigh arteryhas been removed.

FIG. 8 is a series of images showing the probability of tissue necrosis(center) based on a perfusion map of the ischemia leg indicatingischemia rates measured immediately after an operation (left side), anda photograph showing the actual rates of tissue necrosis seven daysafter an operation (right side).

FIG. 9 is a graph showing the experimental results of the dynamics ofthe intensity of ICG fluorescence in blood collected from a living body;and

FIG. 10 is a diagram illustrating an example of the principle of mappingbetween perfusion rates and colors.

BEST MODE

The present invention will be described in detail below.

The present invention provides an analysis apparatus for measuring therate of tissue perfusion by injecting indocyanine green (ICG) into aliving body and measuring variation in concentration, and thenpresenting information about the probability of the necrosis of tissue.

The analysis apparatus includes:

1) an input means for receiving signals from a photodetector, 2) anumerical conversion means for processing the input signals into theintensities of fluorescence with the passage of time in a region ofinterest, 3) a perfusion rate calculation means for calculating therates of perfusion for respective regions of a tissue using thenumerical values, and 4) an output means for outputting the results ofthe calculation.

In the analysis apparatus, the photodetector is a device for detectingfluorescence in a typical infrared ray, visible ray or ultraviolet rayregion. Although not limited to following devices, a photo-detectiondevice, such as a photoresistor, a photovoltaic cell, a photodiode, aphotomultiplier tube (PMT), a phototube, a phototransistor, acharge-coupled device (CCD), a pyroelectric detector, a Golay cell, athermocouple, a thermistor, a complementary metal oxide semiconductor(CMOS) detector or a cryogenic detector, may be used as thephotodetector.

It is preferable to use RSC 232, a parallel port, IEEE 1394 or USB asthe input means, although the invention is not limited thereto.

The numerical conversion means is operably connected to the input means,and consists of a microprocessor, and operation software embeddedtherein. The intensity of fluorescence may be numerically convertedusing a graph, with the intensity of fluorescence being plotted on the yaxis and the time of measurement being plotted on the x axis, as shownin FIGS. 3 and 4, but may be numerically converted using some othermethods.

The calculation means includes a microprocessor, and operation softwareembedded therein and configured to drive an algorithm including acalculation equation for calculating perfusion using the numerical valueof the intensity of fluorescence and the time of measurement, obtainedfrom the numerical conversion means. The microprocessor may be amicroprocessor included in the numerical conversion means, or may be aseparate microprocessor.

The calculation uses the fact that the intensity of fluorescence in anormal tissue decreases exponentially with the passage of time ofmeasurement, and the fact that, in the case where ischemia occurs in atissue that is the target of analysis, the ICG fluorescence particles ofnormal tissue enter at the perfusion rate of an ischemic tissue and theICG fluorescence particles of the ischemic tissue exit at the sameperfusion rate.

In the present invention, the rate of tissue perfusion can be calculatedusing the time at which the intensity of fluorescence in a target tissuefor analysis is maximized. This uses the fact that, in the case whereischemia occurs in the target tissue for analysis, the time at which theintensity of ICG fluorescence in the ischemic tissue is highest is thepoint at which the time-differential value is 0. In the presentinvention, the time is defined as T_(max).

In a specific embodiment of the present invention, T_(max) is obtainedusing the following method. First, a graph (See FIG. 3), in which imagesof the ICG fluorescence of a living body, obtained with the passage oftime after the injection of ICG using the above-described method, wereconverted into numerical values with the highest brightness set to 1,was constructed. Since this graph shows a curve that decreasesexponentially according with time, calculation can be conducted usingthe following equation:

${FI}_{nor} = {A \times {{\mathbb{e}}^{- \frac{t}{\tau}}.}}$

In the above equation, FI_(nor) (Fluorescence Intensity normal) meansthe intensity of ICG fluorescence of normal tissue, ‘A’ means theintensity of fluorescence obtained from ICG images during the firstminute (in the present invention, A is calculated as 1), τ is defined ast_(1/2)/ln 2 through calculation, and t_(1/2) means the time when theintensity of fluorescence of ICG is half the highest value.

The extent of variation in the intensity of ICG fluorescence in anischemic tissue, in which the rate of tissue perfusion is lower thanthat of a normal tissue, can be calculated using the following equationbased on the assumption that “the ICG fluorescence particles of thenormal tissue (FI_(nor)) enter at the rate of perfusion of the ischemictissue (P) and the ICG fluorescence particles of the ischemic tissue(FI_(isc)) exit at the same perfusion rate (P)”:

${FI}_{isc} = {\frac{PA}{P - {1/\tau}}{\left( {{\mathbb{e}}^{- \frac{t}{\tau}} - {\mathbb{e}}^{- {Pt}}} \right).}}$

The variation of ICG dynamics, in case when the perfusion rate of theischemic tissue is lowered, was simulated using the above equation (SeeFIG. 4). Through the simulation, the result, in which T_(max), which isthe time at which the intensity of fluorescence was maximized, increasedas the perfusion rate of a tissue decreased, was obtained (See FIG. 5).

The above relationship between T_(max) and the rates of blood perfusionis expressed by the following Equation:

$\begin{matrix}{{{{- {In}}\;\tau} - \frac{T_{\max}}{\tau}} = {{InP} - {{PT}_{\max}.}}} & (1)\end{matrix}$

The rate of tissue perfusion (P) is calculated based on T_(max) usingEquation 1.

However, the measurement of the perfusion rate of a tissue based onT_(max) is only an embodiment of the present invention, and it isapparent to those skilled in the art that the scope of protection of thepresent invention is not limited by the above embodiment. Accordingly,analysis apparatuses for injecting indocyanine green, measuring theconcentration of indocyanine green in tissue with the passage of time,and measuring the perfusion rate of the tissue using the mechanism ofthe dynamics of fluorescence of indocyanine green in tissue fall withinthe scope of the rights of the present invention, regardless of thealgorithm or calculation equation used therein. Meanwhile, in thepresent invention, the calculated perfusion rates are outputted as aperfusion map wherein the calculated perfusion rates mapped to specificcolors. Although it is preferable to use a color gradient for colorscorresponding to the rates of perfusion, the present invention is notlimited thereto, and a programmer may arbitrarily select a colorrepresentation method. The perfusion map as a means for visualizing therate of tissue perfusion, is outputted through a screen, a printer orthe like, and makes an operator of the perfusion rate analysis apparatusof the present invention perceive the rate of tissue perfusion visuallyand intuitively.

Through the embodiment, a relationship between the perfusion rates,measured using the above-described method, and the probability of futurenecrosis of a tissue due to ischemia was obtained. In a specificembodiment, the case where the rate of tissue perfusion is about 50%/minis indicated by a yellow color in the perfusion map (See the left sideof FIG. 8), and, in the case of this yellow color, the probability ofnecrosis of a tissue was statistically calculated to be in the range of0˜10%, and it may be colored blue in a tissue necrosis probability map(See the center of FIG. 8 Furthermore, the present invention provides atissue perfusion measurement apparatus, comprising a stand configured toallow light to pass therethrough, a light source adjustable to belocated to radiate light onto an ICG-injected living body which isdisposed on the stand, a filter adjustable to be located to filter onlynear infrared ray wavelengths of 800 to 850 nm from fluorescence signalsemitted from the living body under the action of the light source, adetector configured to detect fluorescence light passed through thefilter, and an analysis apparatus operably connected to the detector andconfigured to image light detected by the detector and measure the ratesof tissue perfusion through the analysis of the ICG dynamics with thepassage of time.

With regard to the light source, a Light Emitting Diode (LED) having awavelength of 770+/−20 nm, a light source in which a filter having awavelength of 770+/−20 nm is attached to a white light source, and alaser light source having a wavelength of 770+/−20 nm may be all used asthe light source radiating light for detecting ICG fluorescence.

The above-described filter passes only light having a wavelength rangingfrom 800 to 850 nm therethrough. Using this filter, fluorescencegenerated in a living body itself is not detected by the detector.

The detector detects near infrared rays having wavelengths ranging from800 to 850 nm, which are ICG fluorescence signals. An infrared detectioncamera or a spectrometer may be used as the detector. The detectorcontinuously detects images immediately from the injection of ICG, andtransmits signals, capable of exhibiting an ICG dynamics with thepassage of time, to the analysis apparatus.

In the present invention, when a Region Of Interest (ROI) of a livingbody, in which the perfusion rate is desired to be measured, isdesignated, the intensities of ICG fluorescence with the passage of timeare automatically converted into numerical values from continuous ICGimage data. Using this, the intensities of ICG fluorescence, measured asin FIG. 3, are processed into temporal and spatial information. T_(max),is obtained based on the data, and then the rates of tissue perfusionare analyzed using the simulation data of FIG. 4, the correlationbetween T_(max), of FIG. 5 and the rates of tissue perfusion, and theEquation 1. Furthermore, using the results of the analysis, a tissueperfusion map (See FIG. 7 and the left side of FIG. 8) and a tissuenecrosis rate prediction map (See the center of FIG. 8) are provided.This prediction exhibited a high prediction level in the embodiment ofthe present invention (See the right side of FIG. 8).

In order to measure an ICG dynamics in a living body using the abovemeasuring apparatus, ICG must be injected through an intravenousinjection and variation in the concentration of ICG in tissue with thepassage of time must be detected and analyzed. The variation in theconcentration of ICG in tissue may be measured through the acquisitionof a blood sample, through the direct acquisition of a near infrared rayimage of the tissue, or using a spectroscopic technique.

Furthermore, the present invention provides a method of measuring therate of tissue perfusion, comprising:

1) detecting sequentially concentration of ICG until the concentrationof ICG is sufficiently decreased by injecting the ICG into a livingbody, radiating light onto the living body using a light source, andmeasuring the intensities of fluorescence, generated in the living bodywith the passage of time after the injection, using a photodetector;

2) processing the sequential concentration of ICG in the living bodydetected in the step 2 into numerical data by converting the intensitiesof ICG fluorescence into numerical values for respective regions andrespective times; and

3) calculating the rates of perfusion from the numerical data.

In an embodiment, it is preferred that the method of measuring the ratesof tissue perfusion further comprises the step of outputting thecalculated perfusion rates as a perfusion map. It is preferred that theperfusion map is constructed by painting particular pixels withrespective colors corresponding to the rates of perfusion according to agradient map.

According to another embodiment, the present invention provides a methodof measuring the rates of tissue perfusion, comprising:

1) detecting sequentially concentration of ICG until the concentrationof ICG is sufficiently decreased by injecting the ICG into a livingbody, radiating light onto the living body using a light source, andmeasuring intensities of fluorescence, generated in the living body withthe passage of time, using a photodetector and obtaining an ICG bloodvessel image diagram using the intensities of fluorescence;

2) acquiring T_(max) by analyzing dynamics of the intensities of ICGfluorescence per respective pixel in the ICG blood vessel image diagram;and

3) calculating the rates of perfusion rate based on the T_(max).

In this case, it is preferred that the method of measuring the rates oftissue perfusion further comprises the step of outputting the calculatedperfusion rates as a perfusion map. It is preferred that the perfusionmap is constructed by painting particular pixels with respective colorscorresponding to the rates of perfusion according to a color gradientmap, wherein the rates of perfusion is calculated through the analyzeddata.

Furthermore, the present invention provides a method of predicting therate of tissue necrosis, comprising representing a tissue necrosisprobability map, indicating the probability of necrosis of a tissue,based on the rates of tissue perfusion acquired using theabove-described measurement method, as a result (See the right side ofFIG. 8).

In the measurement method, with regard to the light source, an LEDhaving a wavelength of 770+/−20 nm, a light source in which a filterhaving a wavelength of 770+/−20 nm is attached to a white light source,and a laser light source, having a wavelength of 770+/−20 nm, may all beused as a light source radiating light for detecting ICG fluorescence.Any device using the above-described photo-detection device may be usedas the photodetector. Although the photodetector is not limited, thephotodetector must detect near infrared rays having wavelengths rangingfrom 800 to 850 nm, which are ICG fluorescence signals, and an infrareddetection camera or a spectrometer may be used as the photodetector. Thephotodetector sequentially detects images immediately after an injectionof ICG, and transmits signals, capable of exhibiting an ICG dynamicswith the passage of time, to the analysis apparatus.

Meanwhile, with regard to fluorescence emitted from a living body, it ispreferred that fluorescence generated from a living body itself is notdetected using a filter capable of passing only light having wavelengthsranging from 800 to 850 nm therethrough.

It is preferred that the data processing step and the perfusion rateanalyzing step are performed using a microprocessor and softwareembedded in the microprocessor, or stored in external storage, such as ahard disk drive, an optical drive or a flash memory. This software isnot limited to a specific algorithm, as described above, and may beimplemented through one of various platforms, such as Windows seriesoperating systems, for example, Microsoft Windows XP, 2000, Me, 98 and95, Linux, OS/2, and Unix.

Although the correlation coefficient at the perfusion rate analysis stepis not limited thereto, it is preferred that the correlation coefficientis the time at which the intensity of ICG fluorescence of a targettissue to be analyzed (ischemic tissue) is maximized.

Although the acquisition of T_(max) at the step of calculating perfusionrates from the dynamics of ICG fluorescence intensity of the targettissue (ischemic tissue) does not limit the method of analyzing theperfusion rates, it is preferable to perform calculation using theprinciple in which the time T_(max), at which the differential value ofthe intensity of fluorescence is 0, is proportional to the perfusionrate in the target tissue.

The step 3 may be embodied by the following steps: perfusion rates ofregion of interest (ROI) according to (a) average data of pixels ofparticular regions, (b) respective pixels, or (c) respective ROIsrepresenting perfusion rates; and representing the perfusion rates usinga perfusion map by illustrating the perfusion rates for the ROI withcorresponding colors designated according to the perfusion rates. Theabove process may be performed by calculations using software having anappropriate algorithm and loaded on a microprocessor and a displaythrough an output device of a computer. FIG. 7 shows an example of theperfusion map, which may be acquired by sequentially obtainingfluorescence images with the passage of time after injecting ICG,measuring the perfusion rates through the analysis of the ICGfluorescence dynamics of respective pixels, and painting the pixels withcolors corresponding to the measured perfusion rates. An example ofmatching between the perfusion rates and colors is illustrated in FIG.10.

Although the output device is not limited thereto, it is preferred thatthe output device is a monitor, a printer or a plotter. It is possibleto store data in various graphic formats using an external storagedevice.

The present inventors carried out a surgical operation of removing anartery and vein from the thigh of a leg of a nude mouse, acquired ICGimages using the above-described method, and obtained the T_(max) value.Since the rate of tissue necrosis was greatly affected by the rate oftissue perfusion, whether the ‘precise perfusion rates’ were measuredwas evaluated through the observation of rates of tissue necrosis. As aresult, as shown in FIG. 8, the method of the present invention showedthe significant relationship between measured perfusion values and therates of tissue necrosis. This indicates that measurement can beperformed in the case where the perfusion rate is decreased to0.04˜50%/min (the perfusion rate of a normal leg: 300%/min), unlike themeasurement using Doppler imaging (refer to FIG. 1), that is, acomparative example, in which a subtle difference in the considerablydecreased ‘perfusion rate’ could not be measured.

MODE FOR INVENTION

The present invention will be described in detail below in conjunctionwith embodiments.

However, the following embodiments are only to illustrate the presentinvention, but do not limit the content of the present invention.

COMPARATIVE EXAMPLE 1 Prediction for Tissue Necrosis through DopplerImaging

In FIG. 1, showing the present inventors' experimental data, the threeleft views thereof show data obtained through laser Doppler imaging oneto four days after a surgical operation of removing an artery and veinfrom the thigh of one leg of a mouse so as to construct a bloodperfusion reduction model, and the right view thereof shows the rate ofnecrosis of the leg tissue a week thereafter. With regard to the ratesof tissue necrosis, the case A shows no necrosis, the case B showsnecrosis reaching the center of the sole of the foot, and the case Cshows necrosis reaching the ankle, and the rate of necrosis isproportional to the rate of tissue perfusion (Helisch, A. et al., (2006)Arterioscler. Thromb. Vasc. Biol. 26: 520-526, the case A shows 160% ofnormal perfusion, the case B shows 20% of normal perfusion, and the caseC shows 5% of normal perfusion). Respective pieces of Doppler imagingdata corresponding to the cases did not exhibit any difference in tissueperfusion between the cases.

EXAMPLE 1 Establishment of Method of Measuring Perfusion Using ICG

The present inventors derived an equation through the following steps soas to establish a method of measuring perfusion based on the ICGconcentration dynamics in blood.

FIG. 9 is a graph that is obtained by injecting ICG (1.5 mg/kg, Sigma,USA) into a vein, collecting blood over time, measuring the intensitiesof ICG fluorescence, and converting the intensities into theconcentrations of ICG in blood. The closed squares of the graph indicatethe intensities of ICG fluorescence, while the open squares thereofindicate the concentrations of ICG in blood. FIG. 3 shows variation inICG fluorescence over a period of time from 1 minute to 12 minutes afterthe injection of ICG. That is, in FIG. 3, an image of the ICGfluorescence of a living body acquired with the passage of time isconverted into numerical values, with the highest brightness set to 1,and is represented in the form of a graph. When variation in ICG innormal tissue is formulated using a graph acquired from a normal tissue,as shown in FIG. 3, the graph decreases exponentially with respect totime, and thus the following formulation can be realized:

${{FI}_{nor} = {A \times {\mathbb{e}}^{- \frac{t}{\tau}}}},$Wherein the FI_(nor) (Fluorescence Intensity normal) is the intensity ofICG fluorescence in the normal tissue, A is the intensity offluorescence obtained from an ICG image for a first minute (in thepresent invention, A is calculated as 1), and τ is defined as t_(1/2)/ln2 through calculation. Furthermore, t_(1/2) is the time at which theintensity of ICG fluorescence is half of the highest value.

A simulation was made under the expectation that, when tissue perfusiondecreases, the speed of dispersion of ICG decreases, with the resultthat variation in the intensity of ICG fluorescence differs from that ofnormal tissue. In this case, the ‘perfusion rate’ is ‘the rate at whichblood in a region of interest is exchanged for blood in the otherregions for one minute with respect to the total amount of blood.’ Theunit of the perfusion rate is %/min, and the ‘perfusion rate’ of anormal leg was calculated as 300%/min. The rate of variation in theintensity of ICG fluorescence with the passage of time in an ischemictissue, the perfusion rate of which is lower than in a normal tissue, isexpressed by the following equation on the assumption that “ICGfluorescence particles FI_(nor) in the normal tissue enter at theperfusion rate P of the ischemic tissue, and ICG fluorescence particlesFI_(isc) in the ischemic tissue exit at the same perfusion rate P.”

The following equation is given for FI_(isc) (FI_(ischemia)) as follows:

${FI}_{isc} = {\frac{PA}{P - {1/\tau}}{\left( {{\mathbb{e}}^{- \frac{t}{\tau}} - {\mathbb{e}}^{- {Pt}}} \right).}}$

In this case, the simulation graph of FIG. 4 is obtained by substitutinga perfusion rate of 20-300%/min for P. In this graph, as the perfusionrate decreases, the maximum value of the intensity of ICG fluorescencedecreases, and the time at which the intensity of ICG fluorescence ismaximized is delayed.

An actual ICG fluorescence intensity graph differs from a simulatedgraph. The decrease in the intensity of ICG fluorescence in a normaltissue varies with the state of a living body, with the result thatabsolute comparison is possible only when the fluorescence in anischemic tissue is corrected. As a result, a ‘correlation coefficient’,which is a standard criterion for obtaining ‘perfusion rates’ fromrespective pieces of experimental data, is required, and, in the presentinvention, T_(max) is selected as the correlation coefficient. T_(max)is the time at which the differential value of the ICG fluorescenceintensity dynamics of an ischemic tissue is 0.

Since T_(max), which is the time at which the intensity of ICGfluorescence in an ischemic tissue is highest, is the point at which thetime-differential value is 0, the following equation can be given:

${\frac{\mathbb{d}{{FI}_{isc}\left( T_{\max} \right)}}{\mathbb{d}t} = 0},\mspace{14mu}{and}$$\frac{\mathbb{d}{{FI}_{isc}\left( T_{\max} \right)}}{\mathbb{d}t} = {{\frac{PA}{P - {t/\tau}}\left\lbrack {{{- \frac{1}{\tau}}{\mathbb{e}}^{- \frac{T_{\max}}{\tau}}} + {P\;{\mathbb{e}}^{- {PT}_{\max}}}} \right\rbrack} = 0.}$

These can be summarized as follows:

$\begin{matrix}{{{{- {In}}\;\tau} - \frac{T_{\max}}{\tau}} = {{{In}\; P} - {{PT}_{\max}.}}} & (1)\end{matrix}$

A graph for T_(max) and the ‘perfusion rate’ is shown in FIG. 5.

EXAMPLE 2 Measurement of Perfusion Using Indocyanine Green and theConstruction of Perfusion Map and Tissue Necrosis Probability Map Basedon Correlation Coefficient

The present inventors constructed a blood perfusion reduction model toobtain ICG images in an actual ischemic tissue. A surgical operation ofremoving an artery and vein from one leg of each of 50 nude mice(Charlse liver Japan, Inc.) was carried out, ICG images were acquiredusing the measurement apparatus of FIG. 2 four hours after the surgicaloperation, and the perfusion rates of tissues were obtained using theabove-described method.

The obtained perfusion rates of tissues were arranged in a perfusion map(the left side of FIG. 8). In FIG. 8, a perfusion rate of 300%/mincorresponds to 9 in the perfusion map, 0%/min corresponds to 0.

Since the rate of tissue necrosis is considerably affected by the rateof tissue perfusion, whether a ‘perfusion rate’ has been preciselymeasured can be evaluated through the observation of the rate of tissuenecrosis. The rates of tissue necrosis of 50 laboratory mice wereobserved seven days after operations (the right side of FIG. 8), and therelationship between perfusion rates and the rates of tissue necrosis,which were obtained four hours after the operations, was statisticallyanalyzed using the statistical data. As a result of the analysis, it wasascertained that the probability of tissue necrosis corresponding to aperfusion rate of 50%/min fell within a range of 0˜10%, and theprobability of tissue necrosis corresponding to a perfusion rate rangingfrom 15 to 20%/min fell within a range of 75 to 80%. The presentinventors assigned a red color to the case where the probability oftissue necrosis was 1 and a blue color to the case where the probabilityof tissue necrosis was 0, and constructs a necrosis probability map forthe tissue using a color gradient between the cases (the center of FIG.8). The result, in which the constructed tissue necrosis probability mapcorresponded to the actual rates of tissue necrosis, was acquired.

The result exhibited the fact that the probability of necrosis of tissueincreased as the perfusion rate measured using the method decreased, andthus verified the precision of ‘perfusion rates’ acquired using themethod. This means that it was impossible to measure a subtle differencein considerably decreased ‘perfusion rate’ using the conventionaltechnology, as shown in FIG. 1, but it is possible to performmeasurement using the method of the present invention in the case wherethe perfusion rate is decreased to 0.04-50%/min (the perfusion rate of anormal leg: 300%/min). This coincides with the rate of tissue necrosis,which means that the method of the present invention has an ability topredict the rate of tissue necrosis.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, it is possible tomeasure perfusion rates precisely in a wide range from a perfusion ratedecreased to less than 10% to a perfusion rate increased to the normalperfusion rate, and it is possible to predict the rate of tissuenecrosis based on the perfusion rate. In addition, since it is possibleto measure variation in blood perfusion, the present invention can beused for the diagnosis of blood perfusion rate and the measurement ofvariation in perfusion rate after an operation in a region in which thenecrosis of a tissue does not appear in actual clinical situations.Furthermore, the present invention can be used for a blood vesselendothelial cell function test by comparing an ICG fluorescence dynamicswhen the ICG is injected in a normal blood vessel with an ICGfluorescence dynamics varied by drugs inducing activation of bloodvessel endothelial cells or external pressure.

1. A computer for measuring a rate of tissue perfusion using time-seriesanalysis of fluorescence images of indocyanine green (ICG),comprising: 1) an input module configured to receive signals from aphotodetector, 2) a numerical conversion module configured to processinput signals from the input module into numerical values, said inputsignals comprising intensities of fluorescence over time in a region ofinterest, 3) a rate calculation module configured to calculate rates oftissue perfusion with using the numerical values and an equationrepresenting dynamics of the ICG, and 4) an output module configured tooutput results of the calculation, wherein the equation representingdynamics of the ICG is either${{FI}_{isc} = {{{\frac{PA}{P - {1\text{/}\tau}}\left( {{\mathbb{e}}^{- \frac{t}{\tau}} - {\mathbb{e}}^{- {Pt}}} \right)\mspace{14mu}{or}}\mspace{14mu} - {{In}\;\tau} - \frac{T_{\max}}{\tau}} = {{InP} - {PT}_{\max}}}},$wherein the FI_(isc) (Fluorescence Intensity ischemia) is an intensityof ICG fluorescence of a measurement target tissue, the P is a rate oftissue perfusion, the A is an intensity of fluorescence obtained fromICG image during the first 1 minute, the τ is t_(1/2)/ln2, the t_(1/2)is a time at which the intensity of ICG fluorescence is half of ahighest value and the T_(max) is a time at which the intensity of ICGfluorescence in an ischemic tissue is highest.
 2. An apparatus formeasuring tissue perfusion, comprising: a stand configured to allowlight to pass therethrough, a light source adjustable to be located toradiate light onto an ICG-injected tissue disposed on the stand, afilter adjustable to be located to filter only near infrared raywavelengths of 800 to 850 nm from fluorescence signals emitted from theliving body under the action of the light source, a detector configuredto detect fluorescence light passed through the filter, and the computerof claim 1 operably connected to the detector and configured to imagelight detected by the detector and measure the rates of tissue perfusionthrough the analysis of the ICG dynamics with the passage of time.
 3. Amethod of measuring a rate of tissue perfusion, comprising: 1) detectingsequentially a concentration of ICG until the concentration of ICG isdecreased to a lowest level by injecting the ICG into a living body,radiating light onto the living body using a light source, and measuringintensities of ICG fluorescence generated in the living body with thepassage of time after the injection using a photodetector; 2) processingthe sequential concentration of ICG in the living body detected in thestep 1) into numerical data by converting the intensities of ICGfluorescence into numerical values for respective regions and respectivetimes; and 3) calculating the rates of perfusion from the numericalvalues, wherein the perfusion rates are calculated using the followingequation:${{FI}_{isc} = {{{\frac{PA}{P - {1\text{/}\tau}}\left( {{\mathbb{e}}^{- \frac{t}{\tau}} - {\mathbb{e}}^{- {Pt}}} \right)\mspace{14mu}{or}}\mspace{14mu} - {{In}\;\tau} - \frac{T_{\max}}{\tau}} = {{InP} - {PT}_{\max}}}},$wherein the FI_(isc) (Fluorescence Intensity ischemia) is an intensityof ICG fluorescence of a measurement target tissue, the P is a rate oftissue perfusion, the A is an intensity of fluorescence obtained fromICG image during the first 1 minute, the τ is t_(1/2)/ln2, the t_(1/2)is a time at which the intensity of ICG fluorescence is half of ahighest value and the T_(max) is a time at which the intensity of ICGfluorescence in an ischemic tissue is highest.
 4. The method as setforth in claim 3, further comprising outputting the calculated perfusionrates as a perfusion map.
 5. The method as set forth in claim 4, whereinthe perfusion map is constructed by coloring particular pixels withrespective colors corresponding to the rates of perfusion according to acolor gradient map.
 6. A method of measuring a rate of tissue perfusion,comprising: 1) detecting sequentially a concentration of ICG until theconcentration of ICG is decreased to a lowest level by injecting the ICGinto a living body, radiating light onto the living body using a lightsource, and measuring intensities of fluorescence generated in theliving body with the passage of time using a photodetector and obtainingan ICG blood vessel image diagram using the intensities of fluorescence;2) acquiring T_(max) by analyzing dynamics of the intensities of ICGfluorescence per respective pixel in the ICG blood vessel image diagram;and 3) calculating the rates of perfusion rate based on the T_(max),wherein the perfusion rates are calculated using the following equation:${{{{- {In}}\;\tau} - \frac{T_{\max}}{\tau}} = {{{In}\; P} - {PT}_{\max}}},$wherein the P is a perfusion rate, and the τ is t_(1/2)/ln2, the t_(1/2)is a time at which the intensity of ICG fluorescence is half of ahighest value and the T_(max) is a time at which the intensity of ICGfluorescence in an ischemic tissue is highest.
 7. The method as setforth in claim 6, further comprising outputting the calculated perfusionrates as a perfusion map.
 8. The method as set forth in claim 7, whereinthe perfusion map is constructed by coloring particular pixels withrespective colors corresponding to the rates of perfusion according to acolor gradient map.
 9. A method of predicting a rate of tissue necrosis,comprising outputting a tissue necrosis probability map by indicatingthe probability of necrosis of a tissue based on the rates of tissueperfusion acquired using the method set forth in claim
 3. 10. The methodas set forth in claim 4, wherein the perfusion map is constructed usinga method of acquiring perfusion rates of region of interest (ROI)according to (a) average data of pixels of particular regions, (b)respective pixels, or (c) respective ROIs representing perfusion rates;and representing the perfusion rates using the perfusion map byillustrating the perfusion rates for the ROI with corresponding colorsdesignated according to the perfusion rates.
 11. A method of predictinga rate of tissue necrosis, comprising outputting a tissue necrosisprobability map by indicating the probability of necrosis of a tissuebased on the rates of tissue perfusion acquired using the method setforth in claim
 6. 12. The method as set forth in claim 7, wherein theperfusion map is constructed using a method of acquiring perfusion ratesof region of interest (ROI) according to (a) average data of pixels ofparticular regions, (b) respective pixels, or (c) respective ROIsrepresenting perfusion rates; and representing the perfusion rates usingthe perfusion map by illustrating the perfusion rates for the ROI withcorresponding colors designated according to the perfusion rates.